1. Area of the Art
The invention relates generally to cyanine dyes and specifically to cyanine dye phosphoramidites, their synthesis and methods of use in labeling of oligonucleotides.
2. Description of the Prior Art
Many procedures employed in biomedical research and recombinant DNA technology rely heavily on the use of oligonucleotides as probes, primers, linkers, adapters, and gene fragments. Several of these uses are described in common laboratory manuals, such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et al., Eds., Current Publications, 1993.
Oligonucleotides of a desirable sequence are synthesized by coupling nucleosides through phosphorous-containing covalent linkages. The most commonly used oligonucleotide synthesis method involves reacting a nucleoside with a protected cyanoethyl phosphoramidite monomer in the presence of a weak acid; oxidation of the formed phosphite linkage; and hydrolysis of the cyanoethyl group (Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach, Beaucage, S. L.; Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311).
Many applications, such as automated DNA sequencing and mapping, in situ detection of hybridization, detection of PCR products, and structural studies, require labeled oligonucleotides. While radioactive labels were traditionally used in these applications, recently certain cyanine dyes have proved to be extremely fluorescent and quite useful in the labeling of biomolecules.
Cyanine dyes offer many desirable properties, including safe handling, absorbency at longer wavelengths, high extinction coefficient, relatively high quantum efficiency, small molecular size, ease of chemical manipulation, and reasonable stability to reagents, pH and temperature. Because of a low background fluorescence of biological materials and a high absorbency of cyanine dyes in the longer wavelength portion of the spectrum, cyanine dyes provide excellent signal-to-noise ratios. By synthesizing structural modifications of the chromophore portion of cyanine dyes, different fluorescent labeling reagents absorbing and emitting in a broad spectrum range from 400 to nearly 1100 nm can be obtained. The versatility of functional groups that can be incorporated into cyanine dyes permits control over the solubility of the dye and labeled product, and helps reduce non-specific binding of the labeled materials to irrelevant components in an assay mixture (Waggoner, U.S. Pat. No. 5,569,587 and U.S. Pat. No. 5,627,027).
At present, labeling of oligonucleotides with cyanine dyes is performed by a manual, two-step procedure. First, an oligonucleotide is synthesized and, then, an activated cyanine dye is linked to the 5' end of the synthesized oligonucleotide. Usually, cyanine dyes are activated by an introduction of reactive groups that assist in covalent attachment of cyanine dyes to oligonucleotides (see, for example, U.S. Pat. Nos. 5,569,587 and 5,627,027). This two-step method is slow (4-5 days), tedious, expensive, and often produces undesirable organic by-products.
In an alternative, more convenient, one-step approach, a fluorescent dye is converted into a phosphoramidite and is used in direct labeling of an oligonucleotide during its synthesis. However, currently available phosphoramidites of cyanine dyes are substantially more expensive and less stable than their standard, unmodified counterparts.
U.S. Pat. No. 5,556,959 ('959) discloses the use of carbocyanine phosphoramidites to label synthetic oligonucleotides. The cyanine phosphoramidites of the '959 patent, however, contain protecting groups, such as trityl; 4-O-monomethoxytrityl; 4,4'-O-dimethoxytrityl or acyl. Protecting groups are usually associated with instability during storage and handling, thus making these phosphoramidites less valuable commercially.